The present invention relates to the field of molecular biology and microbiology. More particularly, the invention relates to bioproduction of dodecanoic diacid from cyclododecannone by microbiological means. The cyclododecanone degradation is mediated by a set of enzymes resident on a 10 kb gene cluster, isolated from Rhodococcus ruber SC1. The invention also relates to the bioproduction of lactones, hydroxy acids and diacids from cyclic compounds.
Dodecanedioic acid (DDDA) and other diacids of different chain length are used in nylon manufacturing for production of different variants of nylon fibers. Nylon 6, 12 is a polymer condensed from monomers of hexamethylenediamine (HMD) and DDDA (C12 diacid), which has some different properties from Nylon 6,6 condensed from HMD and adipic acid (C6 diacid). The toughness of the Nylon 6,12 polymer allows it to be used in applications such as toothbrush bristles. Mixed diacids of different chain length (C11, C12 and C13) can also be used as soft segments in Lycra(copyright) or as corrosion inhibitors in automotive coatings.
Traditional ways of diacids synthesis are based on chemical and physical methods which involve harsh conditions such as very high temperatures and pressures. The chemical method to produce 1,12-dodecanoic diacid (DDDA) employs initial air oxidation of cyclododecane to yield a mixture of cyclododecanone (ketone) and cyclododecanol (alcohol), which is then oxidized to produce DDDA. Biocatalytic processes may provide an economically and environmentally more compatible approach towards nylon production. The biochemical route is performed under mild conditions such as ambient temperatures and atmospheric pressures.
Isolation of strains in the cyclododecane degradation pathway has been reported. (J. D. Schumacher and R. M. Fakoussa, Oxidation and cleavage of alicyclic structures using bacterial biocatalysts, DGMK Tagungsber. (1997), 9704 (Proceedings ICCS ""97, Volume 3), 1583-1586. Schumacher et al., have isolated and examined the growth characteristics of Rhodococcus ruber CD1-411 on cyclododecane as sole carbon source (Schumacher et al, supra). The authors, employing biotransformation experiments and using cyclododecane as substrate and several enzyme inhibitors, proposed that Rhodococcus ruber CD1-411 metabolizes the alicyclic compound, cyclododecane, in a way similar to the degradation of cyclohexane. It is suggested that cyclododecane is first hydroxylated to cyclododecanol which is then dehydrogenated to cyclododecanone (J. D. Schumacher, et al , Appl. Microbiol. Biotechnol, 1999, 52:85-90). It is postulated that this alicylic ketone is then subject to a Baeyer-Villiger oxidation yielding the lactone oxacyclotridecan-2-one (lauryl lactone), which is hydrolyzed to 12-hydroxydodecanoic acid (12-hydroxy lauric acid). Only 12-hydroxydodecanoic acid was detected by this method, under these conditions.
In related experiments, using an inhibitor of lactone hydrolysis, tetraethylpyrophosphate, Schumacher et al were able to detect lactone oxacyclotridecan-2-one (J. D. Schumacher, et al, Appl. Microbiol. Biotechnol, 1999, 52:85-90). The 12-hydroxy lauric acid was further converted to DDDA via 12-oxo lauric acid intermediate by a two-step sequential oxidation. The above proposed pathway suggests metabolic steps for the degradation of cyclododecane based on Baeyer-Villiger oxidation process and appearance of the major metabolites in Rhodococcus ruber CD4.
Cyclododecanone monooxygenase has been purified. This enzyme is responsible for the oxidation of cycloketone to the corresponding lactone. In spite of these findings there are no reports which describe other enzymes necessary in the cyclododecane degradation pathway. Additionally, the literature is silent with respect to genes encoding this cyclododecanone monooxygenase and isolation of the gene cluster responsible for the whole metabolic pathway.
The problem to be solved therefore is to provide a facile, environmentally responsible method for the production of dodecanoic diacid and other useful intermediates. Applicants have solved the stated problem by identifying, isolating and cloning a 10 kb nucleic acid fragment from Rhodococcus ruber SC1 which mediates the conversion of cyclododecanone and other cyclic compounds to dodecanoic diacid. Recombinant Escherichia coli hosts with the DNA containing the 10 kb gene cluster conveys on the host the ability to convert cyclododecanone to dodecanoic diacid.
The present invention provides an isolated nucleic acid fragment encoding a dodecanoic diacid synthesizing enzyme selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12; (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 6xc3x97SSC (1 M NaCl), 50% formamide, 1% SDS at 37xc2x0 C., and a wash in 0.1xc3x97SSC at 60 to 65xc2x0 C.; and (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c).
Additionally the invention provides an isolated nucleic acid fragment having about 80% to about 90% identity to the nucleic acid fragment selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.
The invention further provides polypeptides encoded by the nucleic acid sequences of the present invention. Additionally the invention provides chimeric genes comprising the instant nucleic acid sequences operably linked to suitable regulatory elements and transformed host cells comprising the chimeric genes.
In an alternate embodiment the invention provides a method of obtaining a nucleic acid fragment encoding all or a substantial portion of a dodecanoic diacid synthesizing enzyme comprising: (a) probing a genomic library with the nucleic acid fragment of the present invention; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of the present invention under the following conditions; 6xc3x97SSC (1 M NaCl), 50% formamide, 1% SDS at 37xc2x0 C., and a wash in 0.1xc3x97SSC at 60 to 65xc2x0 C.; and (c) optionally sequencing the genomic fragment that comprises the clone identified in step (b), wherein the sequenced genomic fragment encodes all or substantially all of an amino acid sequence encoding a dodecanoic diacid synthesizing enzyme. Similarly the invention provides a method of obtaining a nucleic acid fragment encoding all or a substantial portion a dodecanoic diacid synthesizing enzyme comprising: (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37; and (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a); wherein the amplified insert encodes a portion of an amino acid sequence encoding a dodecanoic diacid synthesizing enzyme.
The invention further provides a method for the production of dodecanedioic acid comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of cyclododecanone whereby dodecanedioic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12 under the control of suitable regulatory sequences.
Similarly the invention provides a method for the production of lauryl lactone comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of cyclododecanone whereby lauryl lactone is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:2, under the control of suitable regulatory sequences.
Additionally the invention provides a method for the production of 12-hydroxy lauric acid comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of lauryl lactone whereby 12-hydroxy lauric acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:4, under the control of suitable regulatory sequences.
In another embodiment the invention provides a method for the production of 12-oxo lauric acid comprising: contacting a transformed host cell under suitable growth conditions with an effective amount 12-hydroxy lauric acid whereby 12-oxo lauric acid acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:10, under the control of suitable regulatory sequences.
In an alternate embodiment the invention provides a method for the production of dodecanedioic acid comprising: contacting a transformed host cell under suitable growth conditions with an effective amount 12-oxo lauric acid whereby dodecanedioic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:12, under the control of suitable regulatory sequences.
The invention further provides an isolated nucleic acid fragment encoding a dodecanedioic acid synthesizing enzyme selected from the group consisting of: (a) an isolated nucleic acid molecule as set forth in SEQ ID NO:13; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 6xc3x97SSC (1 M NaCl), 50% formamide, 1% SDS at 37xc2x0 C., and a wash in 0.1xc3x97SSC at 60 to 65xc2x0 C.; and (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
Additionally the invention provides a Rhodococcus ruber comprising endogenous genes encoding the proteins as set forth by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12, said Rhodococcus ruber SC1 having the ability to convert cyclododecanone to dodecanedioic acid under suitable growth conditions.
In another embodiment the invention provides a method for the production of hydroxy acids comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of a cyclic ketone selected from the group consisting of C6, C10, C11, C12, C13, and C15 cyclic ketones whereby the corresponding hydroxy acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:2, and SEQ ID NO:4 under the control of suitable regulatory sequences.